Claudin-4 binding peptides, compositions and methods of use

ABSTRACT

The disclosure provides methods and compositions useful for treating claudin-4 associated disorders including cell proliferative disorders. The disclosure also provide claudin-family binding peptides useful in the methods of the disclosure.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35U.S.C. §371 and claims priority to International Application No.PCT/US2009/042221, filed Apr. 30, 2009, which application claimspriority to U.S. Provisional Application No. 61/049,011, filed Apr. 30,2008, the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The disclosure relates to peptides that bind claudin familypolypeptides, compositions and methods of use thereof.

BACKGROUND

Tight junctions or zona occludens assist in providing a regulatedbarrier between the intercellular spaces within sheets of epithelial orendothelial cells. Such tight junctions are an important aspect of thenormal development of tissues such as the skin and mucosal membranes.These junctions may assist in suppressing the formation and spread oftumors. Inadequate or improperly regulated epithelial or endothelialbarrier function contributes to the initiation, maintenance, andexacerbation of inflammation in tissues such as the gut, lungs, andother mucosal linings. Tight junctions separate the apical andbasolateral regions of these cells' membranes, allowing theestablishment of different physiological environments on the oppositesides of a cell sheet, such as the different physiological environmentsrequired for transport of materials across the intestinal epithelium.

SUMMARY

Claudin-4 (CLDN4) is a tight junction transmembrane protein, and is animportant protein in establishing the transepithelial electricalresistance in the mucosal epithelium barrier. It also is the receptorfor the Clostridium perfringens enterotoxin (CPE), and has been found tobe highly expressed in a variety of solid tumors. It is a component ofmucosal immune surveillance by specialized epithelial M cells. Thedisclosure demonstrates that CLDN4 is a therapeutic target, whether forintentionally disrupting the epithelial barrier, targeting tumors, ortargeted delivery of mucosal vaccines or other payloads. The disclosureprovides a plurality of peptides specific for binding to the secondextracellular loop 2 (Ecl2) of CLDN4. The peptides of the disclosureshow similar affinity for CLDN4 as the CPE protein, but due to theirsmall size are likely to be less immunogenic.

The disclosure provides a substantially purified peptide comprising fromabout 10-15 amino acids and containing a tyrosine or tryptophanseparated by 3-4 amino acids followed by 1 or 2 tyrosines and a leucine.The disclosure provides a claudin family polypeptide binding peptidecomprising about 8 to 30 amino acids and comprising the amino acidsequence (Y/W)(Xaa)_(3 or 4) YYXaaL (SEQ ID NO:1) wherein Xaa is anyamino acid. The disclosure provides a peptide comprising a sequence ofbetween 10-30 amino acids containing a sequence of (Y/W)(Xaa)_(3 or 4)YYXaaL (SEQ ID NO:1). In one embodiment, the peptide comprises at leastone D-amino acid. In one embodiment, the peptide comprises a sequenceXaaXaaXaaXaa(Y/W)(Xaa)_(3 or 4) YYXaaL (SEQ ID NO:2). In anotherembodiment, the peptide comprises a sequenceXaaXaa(Y/W)(Xaa)_(3 or 4)Y(Y/Xaa)(L/I)XaaXaa (SEQ ID NO:3). In specificembodiment, the peptide is selected from the group consisting of:SLDAGQYVLVMKANSSYSGNYPYSILFQKF (SEQ ID NO:4), NSSYSGNYPYSILFQKF (SEQ IDNO:5), SSYSGNYPYSIL (SEQ ID NO:6), NSSYSGNYYSIL (SEQ ID NO:7),ASNSSYSGNYSIL (SEQ ID NO:8), SPWSEPAYTLAP (SEQ ID NO:9), andAPWTEHSYYLSL (SEQ ID NO:10). In one embodiment, the peptide binds to aclaudin family polypeptide. In a further embodiment, the peptide bindsto a claudin-4.

The disclosure also provides fusion peptides or polypeptide comprising abinding peptide as set forth above linked to a small molecule,nanostructure, peptide or polypeptide of interest. In one embodiment,the polypeptide or peptide of interest is a nanoparticle, a vaccine, asmall molecule drug, an antibody, or an antigenic composition.

The disclosure also provides retroinverso peptide of the disclosure.

The disclosure also provides a pharmaceutical composition comprising apeptide of the disclosure in a pharmaceutically acceptable carrier.

The disclosure provides a method of modulating inflammation, asthma,allergy, cell proliferative disorders, metastasis of cancer cells, iontransport disorders such as magnesium transport defects in the kidney,inflammatory bowel disease, Clostridium perfringens enterotoxin (CPE)infection, myelin sheath formation disorders such as multiple sclerosis(MS), autoimmune encephalomyelitis, optic neuritis, and progressivemultifocal leukoencephalopathy (PML) in a subject, comprisingadministering a peptide of the disclosure either alone, as a fusion orcombination with nanoparticle, vaccine, antigen, peptide, polypeptide,small molecule or the like in a pharmaceutically acceptable carrier. Inone embodiment, the allergen is selected from the group consisting ofdust mite allergens, pollen, food allergens, drugs like penicillin, andthe like.

The disclosure also provides a method of targeting a therapeutic ordiagnostic to mucosal M cells comprising linking a therapeutic moiety ordiagnostic to a binding peptide of the disclosure to generate a fusionconstruct and contacting a mucosal M cell with the fusion construct. Inone embodiment, the mucosal M cell is in vivo. In another aspect, thetherapeutic moiety is a cytotoxic drug, an immunopotentiating drug, aninhibitory nucleic acid molecule, a peptide, a polypeptide or apeptidomimetic.

Also provided by the disclosure is a vaccine comprising a bindingpeptide of the disclosure linked to an immunogenic molecule. In oneembodiment, the vaccine comprises a fusion polypeptide comprising thebinding peptide and an immunogenic molecule. In another embodiment, theimmunogenic molecule is an HA antigen.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows claudin-4 Ecl2 only does not bind to Cpe30. The Claudin-4Ecl2 domain was synthesized as a biotinylated peptide as described inTable 1. Biotin-Ecl2 peptides were immobilized to Streptavidin (SA)sensor chips at three different configurations; B-Ecl2 was a linearstructure, B-Ecl2-B was tethered to streptavidin by biotins at bothN-and C-termini, and B-lariat-Ecl2 contained a loop via disulfide bondformed between two cysteines flanking the Ecl2 loop. The ligands wereimmobilized at 500 RU, and tested against Cpe30 peptide as the analyte.The control channel was treated the same as the assay channel, butwithout B-peptide. The sensorgrams with 5 μM of Cpe30 are shown.

FIG. 2A-C shows Ecl2 with downstream transmembrane domain is sufficientenough to express optimal binding activity to Cpe30. Biotin-Cpe30 wasimmobilized on an SA chip as the ligand, and a series of Cldn-4 deletionmutant proteins fused to GST were utilized as analytes. (A) Theschematic diagram of full-length claudin-4 and its mutants, with thestructural domains labeled above. (B) The SDS-PAGE analysis of purifiedGST-Cldn-4 mutant fusions. (C) Overlay of the sensorgrams from differentanalytes at medium concentration. GST protein only did not bind Cpe30.Specific binding was identified by subtraction of the control channelfrom the assay channel; this processing was applied to the rest ofexperiments in the study.

FIG. 3 shows binding kinetics of peptides to GST.Cldn4-Ecl2 mutantR4Biotinylated peptides shown in the figure were immobilized to an SAchip as the ligands, the GST-Cldn4.R4 was used as the analyte from 10 nMto 100 nM to measure the kinetics of binding. The processed specificbinding sensorgrams are presented for each ligand. The 50 nM analyteconcentration was repeated twice to confirm reproducibility. The bindingkinetics was analyzed by “BIAevalution 3.1” software with 1:1 (langmuir)binding mode, and the binding constants were summarized in the table.CC4P-2 peptide had no specific binding to GST.Cldn4-Ecl2, as the assayand control channels responded similarly to the different concentrationsof analyte.

FIG. 4 shows Cpe30 mutant MT2 exhibits specific binding to GST-Cldn4.R4.Cpe30 was reduced to shorter 12 amino acid peptides as indicated inTable 1 (Cpe30MT1 through MT3), and tested against GST-Clnd-4.R4 asdescribed for FIG. 3. Cpe30 MT2 was found to specifically interact withCldn4 Ecl2; the sensorgrams are shown here. Cpe30 MT1 and MT3 did notbind Cldn4 Ecl2.

FIG. 5 shows claudin-4 Ecl2 binding motif of peptide ligands. The Cpe17(SEQ ID NO:5) sequence was aligned with several Ecl2-binding peptidesfound to have significant affinity (CC4P-13: SEQ ID NO:10; CC4P-5: SEQID NO: 9; cPE30mt2: SEQ ID NO: 7; and Binding Motif: SEQ ID NO: 2). Thestructure-based alignment was performed according to the crystalstructure of C-terminus of Cpe. A common binding motif was deduced, withthe underlined tyrosine or tryptophan constituting a structuralrequirement for docking of peptide into the Ecl2 cleft.

FIG. 6A-B shows Cpe30 in recombinant influenza hemagglutinin (HA)retains binding activity to Cldn-4 Ecl2. C-terminally His taggedHA-ts-Cpe30 and HA (shown in (A) were expressed in Baculovirus andpurified to homogeneity. HA-specific antibody (H36) was immobilized to aCM5 chip by amine coupling, which was used to capture HA-ts-Cpe30 or HAprotein as the ligand. Cldn-4 Ecl2.R4 was used as the analyte. (B) Theoverlay of sensorgrams of HA-is-Cpe30 with different concentrations ofCldn-4.R4 show the specific interaction. HA protein without Cpe30 showedno binding to Cldn-4 Ecl2; the figure shows that the assay and controlchannels responded similarly at different concentrations of analyte.

FIG. 7 shows bead uptake by M cells using claudin 4-targeting peptide. Asuspension of 10⁹ fluorescent microbeads (0.2 micron) coated withtargeting peptide Cpe30 were instilled into nasal passages, and 10minutes later the NALT was dissected for bead counting. Shown are UEA-1+M cells (green) in whole mount NALT; BALB/c take up Cpe30-coated beads(red) in large numbers.

FIG. 8 shows analysis of bead uptake by M cells in NALT. Counts areshown as beads per 1600 sq. micron area (40×40 micron), from multiplemeasurements from confocal images from at least three mice per group.Low uptake was seen for beads with no peptide and beads with controlpeptide. High uptake was measured for beads coated with Cpe30 peptide.

FIG. 9 shows a vector map of the HA-ts-Cpe30-HT expression construct.

FIG. 10 shows recombinant vaccine antigen production. A truncatedinfluenza hemagglutinin (HA) was produced in baculovirus cultures as HA,HA plus the claudin 4 targeting Cpe30 fusion (HA-cpe), or as HA-cpe withthe addition of a trimerization peptide and spacer peptides between theHA and the C-terminal cpe30 peptide (HA-ts-cpe) as shown in FIG. 3. Notethe stabilization of the high molecular weight trimer by thetrimerization peptide.

FIG. 11 shows antibody titers in Intestinal Contents (IC) from mucosalimmunization. Mice given i.n. 2 ug HA or HA-ts-Cpe30 (3 weekly doses,plus 1 ug cholera toxin with first dose) were tested after four weeks.Serum IgG levels were the same in both groups; titrations fromindividual animals here shows much higher IgA response in intestinalcontents of mice immunized with HA-is-Cpe30 as compared withHA-immunized or naïve animals.

FIG. 12 shows mucosal and serum titers from mucosal immunization.Targeted HA-Cpe30 conjugate (HAcpe) versus HA (with control peptide: ascrambled peptide sequence) as described in the text was used forintranasal immunization. Both intestinal and serum IgA and IgG titerswere higher in the M cell targeted vaccine. Bars show median endpointtiter values. Significant differences between HA and HACpe groups werecalculated for IgA titers in IC (p<0.01) and serum (p<0.05) byMann-Whitney two-tailed test.

FIG. 13A-B shows endpoint titers using HACpe plus HA-flgn. Here, micewere given (A) intranasal HA-control peptide conjugate (10 ug) plus freeflagellin (5 ug first dose only), versus (B) HA-Cpe30 conjugate (10 ug)plus recombinant HA-flagellin (5 ug, 3 doses). IC and serum IgA titersshown. (*, p<0.03 by one-tailed Mann-Whitney test).

FIG. 14 shows SPR analysis of serum from immunized mice. Equal dilutionsof serum from orally immunized mice given HA or HA-CPE30 plus choleratoxin adjuvant. Individual mice are shown here. The HA-CPE30 immunizedserum shows higher binding response.

FIG. 15A-C shows in vitro uptake of HA-HT (A) and HA-CPE (B) PLGAnanoparticles: HA-CPE nanoparticles given to GFP-Claudin4 transfectantsare readily endocytosed by one hour and colocalize internally withGFP-Claudin4 (bright spots), while HA-HT nanoparticles are not taken up.GFP-Claudin4 (green), Fluorescent nanoparticles (red), nuclei (blue).(C) shows a graph setting out the particle uptake data for PLGAnanoparticles.

FIG. 16A-B shows uptake of nanoparticles. Fluorescent particles weregiven either (A) intranasally (NALT) or (B) as oral gavage (Peyer'spatch), and tissues were then processed for image analysis. Arrowsindicate nanoparticles taken into follicles, though in some cases theyare still evident within M cells (green).

FIG. 17A-B shows uptake of Cpe conjugated particles compared to non-Cpeconjugated particles. Fluorescent nanoparticles were given as anintranasal suspension for 1-2 minutes or intragastric gavage for 4hours. NALT (A) and Peyer's patch (B) were dissected, fixed and wholemounts were analyzed for fluorescent nanoparticles taken into thelymphoid follicles, measured by particles per circular area (7850 squaremicrons). P values for one tailed Mann-Whitney test shown; data combinedfrom two independent experiments. Note that enhanced uptake mediated byCPE targeting is more evident for Peyer's patch compared to NALT. Forcomparison, uptake of fluorescent beads conjugated to streptavidin, thencoated with biotinylated Cpe30 peptide bound were followed, specificuptake is also evident for these particles as compared to beads coatedwith a control peptide.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polynucleotide”includes a plurality of such polynucleotides and reference to “thepeptide” includes reference to one or more peptides, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

Any publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The Claudin polypeptides are a group of “tetraspan” polypeptides,polypeptides having four membrane-spanning or transmembrane domains thatare associated with cellular tight junctions. Claudin familypolypeptides are expressed in epithelial cells and/or endothelial cellsthroughout development, with individual members of the Claudinpolypeptide family being expressed in different tissues. Thephysiological functions associated with a particular Claudin polypeptideare related to the functions performed by the particular tissue(s) inwhich it is expressed.

Because of their roles in tight junction formation, epithelial andendothelial barrier function, ion transport, and viral protein,enterotoxin, or allergen binding, Claudin polypeptides are associatedwith conditions involving unregulated or improperly regulated transportacross the epithelium or endothelium such as inflammation, asthma,allergy, metastasis of cancer cells, and ion transport disorders such asmagnesium transport defects in the kidney. In addition, because aClaudin polypeptide expressed in neural cells has been shown to berequired for formation of the myelin sheath in oligodendrocytes, Claudinpolypeptides are associated with demyelination conditions such asmultiple sclerosis (MS), autoimmune encephalomyelitis, optic neuritis,progressive multifocal leukoencephalopathy (PML), and the like.

Characteristics and activities of the Claudin polypeptide family aredescribed further in the following references: Fujitaab K et al., 2000,Clostridium perfringens enterotoxin binds to the second extracellularloop of claudin-3, a tight junction integral membrane protein, FEBSLett. 476: 258-261; Kinugasa T et al., 2000, Claudins regulate theintestinal barrier in response to immune mediators, Gastroenterology118: 1001-1011; Tsukita S and Furuse M, 2000, Pores in the wall:claudins constitute tight junction strands containing aqueous pores, J.Cell Biol. 149: 13-16; Bronstein J M et al., 2000, Involvement ofOSP/claudin-11 in oligodendrocyte membrane interactions: role in biologyand disease, J Neurosci Res. 59: 706-711; Itoh M et al., 1999, Directbinding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3,with the COOH termini of claudins, J Cell Biol. 147: 1351-1363; Furuse Met al., 1999, Manner of interaction of heterogeneous claudin specieswithin and between tight junction strands, J Cell Biol. 147: 891-903;Morita K et al., 1999, Endothelial claudin: claudin-5/TMVCF constitutestight junction strands in endothelial cells, J Cell Biol. 147: 185-194;Kubota K et al., 1999, Ca(2+)-independent cell-adhesion activity ofclaudins, a family of integral membrane proteins localized at tightjunctions, Curr Biol. 9: 1035-1038; Wan H et al., 1999, Der p 1facilitates transepithelial allergen delivery by disruption of tightjunctions, J Clin Invest. 104: 123-133; Simon D B et al., 1999,Paracellin-1, a renal tight junction protein required for paracellularMg²⁺ resorption, Science 285: 103-106; Morita K et al., 1999, Claudinmultigene family encoding four-transmembrane domain protein componentsof tight junction strands, Proc Natl Acad Sci USA. 96: 511-516; Furuse Met al., 1998, A single gene product, claudin-1 or -2, reconstitutestight junction strands and recruits occludin in fibroblasts, J CellBiol. 143: 391-401; Furuse M et al., 1998, Claudin-1 and -2: novelintegral membrane proteins localizing at tight junctions with nosequence similarity to occludin, J Cell Biol. 141: 1539-1550; all ofwhich are incorporated by reference herein.

The following shows the coding sequence of human claudin-4, otherorthologs and homologs can be identified by performing a routine BLASTsearch of publicly available databases:

(SEQ ID NO: 11) 1atggcctcca tggggctaca ggtaatgggc atcgcgctgg ccgtcctggg ctggctggcc 61gtcatgctgt gctgcgcgct gcccatgtgg cgcgtgacgg ccttcatcgg cagcaacatt 121gtcacctcgc agaccatctg ggagggccta tggatgaact gcgtggtgca gagcaccggc 181cagatgcagt gcaaggtgta cgactcgctg ctggcactgc cgcaggacct gcaggcggcc 241cgcgccctcg tcatcatcag catcatcgtg gctgctctgg gcgtgctgct gtccgtggtg 301gggggcaagt gtaccaactg cctggaggat gaaagcgcca aggccaagac catgatcgtg 361gcgggcgtgg tgttcctgtt ggccggcctt atggtgatag tgccggtgtc ctggacggcc 421cacaacatca tccaagactt ctacaatccg ctggtggcct ccgggcagaa gcgggagatg 481ggtgcctcgc tctacgtcgg ctgggccgcc tccggcctgc tgctccttgg cggggggctg 541ctttgctgca actgtccacc ccgcacagac aagccttact ccgccaagta ttctgctgcc 601cgctctgctg ctgccagcaa ctacgtgtaa 

The following shows the amino acid sequence of claudin-4,

(SEQ ID NO: 12) MASMGLQVMGIALAVLGWLAVMLCCALPMWRVTAFIGSNIVTSQTIWEGLWMNCVVQSTGQMQCKVYDSLLALPQDLQAARALVIISIIVAALGVLLSVVGGKCTNCLEDESAKAKTMIVAGVVFLLAGLMVIVPVSWTAHNIIQDFYNPLVASGQKREMGASLYVGWAASGLLLLGGGLLCCNCPPRTDKPYSAKYSAA RSAAASNYV 

As used herein, “Claudin polypeptides” include human Claudin-4 (SEQ IDNO:12) and species homologues and variants and fragments of theseClaudin polypeptides. Claudin polypeptides have biological activitiesand functions that are consistent with those of the other Claudin familypolypeptides. Polypeptides of the Claudin family are expressed in celltypes including epithelial and endothelial cells throughout development.Typical biological activities or functions associated with this familyof polypeptides are tight junction formation, epithelial or endothelialbarrier function, ion transport, viral protein binding, homotypic orheterotypic binding, and binding PDZ domain binding. Polypeptides havingtight junction formation activity bind to othertight-junction-associated molecules to form tight junction structuresthat regulate epithelial or endothelial barrier function andparacellular transport. The tight junction formation activity isassociated with the extracellular loops and, at least under certainconditions, with the cytoplasmic tail domain of Claudin polypeptides.Thus, for uses requiring tight junction formation activity suchpolypeptides include those having the extracellular loop domains andexhibiting tight junction formation activities such as epithelial orendothelial barrier function, paracellular ion transport, or viralprotein binding. The tight junction formation activity of humanClaudin-4 and other Claudin family polypeptides may be determined, forexample, by introducing Claudin polypeptides into cells that do notnormally form tight junctions, such a L fibroblasts, along with occludinor any other polypeptide that the Claudin polypeptide needs to interactwith in the formation of tight junctions, then visualizing the resultingtight junction structures by electron microscopy or immunofluorescencemethods (see, for example, Furuse et al., J Cell Biol. 143: 391-401,1998). Alternatively, the paracellular ion transport activity of humanClaudin-4 and other Claudin family polypeptides may be assayed byelectrophysiology or through the use of luminescent ion indicatormolecules such as aequorin. As described more fully below, suchtechniques can be used to assess the activity of Claudin-4 bindingpeptides of the disclosure.

The term “human Claudin polypeptide activity,” as used herein, includesany one or more of the following: tight junction formation, epithelialor endothelial barrier function, and ion transport activity; homotypicbinding, heterotypic binding, viral protein binding, enterotoxinbinding, and PDZ domain binding activity; as well as the ex vivo and invivo activities of Claudin polypeptides. The degree to which Claudinpolypeptides and fragments and other derivatives of these polypeptidesexhibit these activities can be determined by standard assay methods.Exemplary assays are disclosed herein; those of skill in the art willappreciate that other, similar types of assays can be used to measurethe biological activities of Claudin polypeptides and other Claudinfamily members.

One aspect of the biological activity of Claudin polypeptides includinghuman Claudin-4 is the ability of members of this polypeptide family tobind particular binding partners such homotypic and heterotypicpolypeptides, viral proteins, enterotoxins, and PDZ-domain-containingpolypeptides, with the extracellular loop domains binding, for example,to homotypic polypeptides, and the cytoplasmic tail domain binding toPDZ-domain-containing polypeptides.

The term “binding partner,” as used herein, includes peptides thatinteracts with a Claudin-4 polypeptide through contact or proximitybetween particular portions of the binding partner and the Claudin-4polypeptide. The interactions between Claudin polypeptides and bindingpartners are involved in mediating interactions between adjacentepithelial cells, and interactions between adjacent endothelial cells.Artificial binding peptide or domains (including soluble domains) of thedisclosure either alone or in a fusion construct or polypeptide (e.g.,fused to an immunoglobulin Fc domain, an immunogenic molecule, ananoparticle or the like), is expected to disrupt the binding of Claudinpolypeptides to their normal binding partners or facilitate uptake by adesired cell expressing claudin-4.

Polypeptides of the Claudin family are involved in epithelial orendothelial barrier function and transport diseases or conditions thatshare as a common feature of abnormal tight junction formation orimproperly regulated tight junction function (i.e. abnormal epithelialor endothelial barrier function) in their etiology. More specifically,the following conditions involving epithelial or endothelial barrierfunction and/or binding to Claudin polypeptides are those that are knownor are likely to involve the biological activities of Claudinpolypeptides: inflammation (e.g., psoriasis and other inflammatorydermatoses), asthma, allergy, cell proliferative disorders (e.g.,hyperproliferative skin disorders including skin cancer), metastasis ofcancer cells, ion transport disorders such as magnesium transportdefects in the kidney, inflammatory bowel disease, and exposure toClostridium perfringens enterotoxin (CPE). In addition, because aClaudin polypeptide expressed in neural cells has been shown to berequired for formation of the myelin sheath in oligodendrocytes, Claudinpolypeptides are associated with demyelination conditions such asmultiple sclerosis (MS), autoimmune encephalomyelitis, optic neuritis,and progressive multifocal leukoencephalopathy (PML). Also, diseasesthat are promoted by one or more of the conditions above may involveClaudin polypeptides, directly or indirectly. For example,susceptibility to sudden infant death syndrome (SIDS) has beenassociated with exposure to CPE. Blocking or inhibiting the interactionsbetween a claudin-4 polypeptide and their substrates, ligands, receptorsand or other interacting polypeptides is provided by the disclosure andprovides methods for treating or ameliorating these diseases andconditions through the use of inhibitors of human Claudin-4 activity.Examples of such inhibitors or antagonists are described in more detailbelow. Additional uses for Claudin binding peptides include diagnosticreagents for epithelial or endothelial transport diseases; researchreagents for investigation or as a carrier or targeting molecule for thedelivery of therapeutic agents or diagnostic agents, particularly inview of the role of Claudins in the tight junctions.

The disclosure also provides compositions and methods for mucosaldelivery of therapeutics, diagnostics and biologically active peptidesand proteins. For example, a therapeutic moiety (e.g., an immunogenicpolypeptide, small molecule drug or diagnostic agent) can be linked to abinding peptide of the disclosure and delivered to a cell or subject,wherein the binding peptide selectively binds to a claudin polypeptide(e.g., a claudin-4 polypeptide). Because claudins are normally expressedon endothelial and epithelial cells, the binding peptide will interactwith such cells in mucosal tissues thereby delivery any linked payloadto the tissue or cell. Useful agents that can be linked to a bindingpeptide of the disclosure include, for example, tissue plasminogenactivator (TPA), epidermal growth factor (EGF), fibroblast growth factor(FGF-acidic or basic), platelet derived growth factor (PDGF),transforming growth factor (TGF-alpha or beta), vasoactive intestinalpeptide, tumor necrosis factor (TNF), hypothalmic releasing factors,prolactin, thyroid stimulating hormone (TSH), adrenocorticotropichormone (ACTH), parathyroid hormone (PTH), follicle stimulating hormone(FSF), luteinizing hormone releasing hormone (LHRH), endorphins,glucagon, calcitonin, oxytocin, carbetocin, aldoetecone, enkaphalins,somatostin, somatotropin, somatomedin, gonadotrophin, estrogen,progesterone, testosterone, alpha-melanocyte stimulating hormone,non-naturally occurring opiods, lidocaine, ketoprofen, sufentainil,terbutaline, droperidol, scopolamine, gonadorelin, ciclopirox, olamine,buspirone, calcitonin, cromolyn sodium or midazolam, cyclosporin,lisinopril, captopril, delapril, cimetidine, ranitidine, famotidine,superoxide dismutase, asparaginase, arginase, arginine deaminease,adenosine deaminase ribonuclease, trypsin, chemotrypsin, and papain.Additional examples of useful peptides include, but are not limited to,bombesin, substance P, vasopressin, alpha-globulins, transferrin,fibrinogen, beta-lipoproteins, beta-globulins, prothrombin,ceruloplasmin, alpha₂-glycoproteins, alpha₂-globulins, fetuin,alpha1-lipoproteins, alpha1-globulins, albumin, prealbumin, and otherbioactive proteins and recombinant protein products.

The disclosure also provides methods and compositions for providingmucosal delivery of specific, biologically active peptide, protein orsmall molecule therapeutics to treat (i.e., to eliminate, or reduce theoccurrence or severity of symptoms of) an existing disease or condition,or to prevent onset of a disease or condition in a subject identified tobe at risk for the subject disease or condition. Biologically activemolecules that are useful include, but are not limited to, cytokines;immunopotentiating agents; growth factors; hormones; hematopoietics;antiinfective agents; antidementia agents; antiviral agents; antitumoralagents; antipyretics; analgesics; antiinflammatory agents; antiulceragents; antiallergic agents; antidepressants; psychotropic agents;cardiotonics; antiarrythmic agents; vasodilators; antihypertensiveagents such as hypotensive diuretics; antidiabetic agents;anticoagulants; cholesterol lowering agents; therapeutic agents forosteoporosis; hormones; antibiotics; vaccines; and the like. Exemplaryhormones include androgens, estrogens, prostaglandins, somatotropins,gonadotropins, interleukins, steroids and cytokines.

Furthermore, vaccines which may be administered within the methods andcompositions of the disclosure include bacterial and viral vaccines,such as vaccines for hepatitis, influenza, Dengue, rotavirus, vibrio,SARS, respiratory syncytial virus (RSV), parainfluenza virus (PIV),tuberculosis, canary pox, chicken pox, measles, mumps, rubella,pneumonia, and human immunodeficiency virus (HIV). For example, HAantigens can be linked to a binding peptide of the disclosure and usedfor immunization. Bacterial toxoids can be used with the methods andcompositions disclosure including, for example, diphtheria, tetanus,pseudonomas and mycobactrium tuberculosis.

Conventional vaccine development has been mainly based on eitherinfection with an attenuated pathogen, or direct subcutaneous orintramuscular injection of inert antigens from the pathogen. Theattenuated pathogen, if available, provides a persistent stimulussimilar to the pathogenic infection. Here, an effective immune responsemay be generated in the regional site most appropriate for protectiveimmunity, but attenuated strains are often not available or they riskcausing the infectious disease itself. By contrast, direct injection ofantigen predominantly induces a systemic IgG response. While this may beadequate in many cases, it is acknowledged to be less effective inproviding regional immunity.

Thus, in mucosal tissues, secretory IgA responses provide the bestprotective response. For respiratory pathogens, secretory IgA is ofobvious benefit, as it is secreted at very high levels onto mucosalsurfaces, where it can neutralize viral pathogens and prevent eitherdirect infection of epithelial cells or entry across the epithelialbarrier. However, immunization for mucosal responses has additionalbenefits to the patient; while systemic immunization can provide onlysystemic (serum) IgG responses, mucosal immunization induces both serumIgG and mucosal IgA responses. Moreover, IgA:virus immune complexes canbe transcytosed by the pIgR intact across the mucosal epithelium intothe intestinal lumen for eventual elimination, thereby potentiallyproviding both protection from mucosal infections and active eliminationof infectious particles that are already in the body, as in the case ofblood-borne infections.

Surprisingly, vaccine development efforts have not yet taken advantageof the additional practical advantages of mucosal immunization.Administration of vaccines targeted to the mucosal immune tissues couldbe done relatively cheaply and easily; with oral or intranasal deliverynot requiring needles for injections, trained medical staff, andspecialized equipment. This principle has been recognized in many mousestudies using methods to target delivery of vaccine antigens to mucosalM cells in the follicle epithelium overlying lymphoid tissues such asPeyer's Patches. M cells are specialized for particle transcytosis fromthe intestinal lumen to the lymphoid follicle, but to date the targetingof vaccines to M cells has relied only on targets unique to mouse andnot present on human M cells. What is needed therefore is a method fortargeting vaccine delivery to the mucosal immune system that usestargets shared by both mouse and human.

For example, M cells are specialized epithelial cells found mainlyoverlying lymphoid follicles such as Nasopharyngeal Associated LymphoidTissue (NALT) in the mouse, tonsils in the human, and Peyer's Patches inboth species. M cells are specialized for antigen and particle uptakeand transcytosis to the basolateral side of the Peyer's Patch FollicleAssociated Epithelium (PPFAE), or NALT epithelium, where waiting antigenpresenting cells take up the antigens for presentation to folliclelymphocytes and stimulate IgA responses. The redistribution of cldn4 inM cells in vivo from tight junctions to the cytoplasm in both mouse andhuman Peyer's Patches shows that this transmembrane protein participatesin particle transcytosis. In support of this hypothesis, lymphotoxin/TNFtreatment of the human intestinal epithelium cell line Caco-2BBe alsoinduced redistribution of cldn4 and that many of the bacteria were foundwithin cldn4-positive vesicles when these cells were allowed toendocytose fluorescently labeled bacterial particles. These resultsimplicate cldn4 as a component of M cell transcytosis. As demonstratedherein, the targeting of extracellular domains of cldn4 mediateefficient delivery of vaccine antigens to mucosal lymphoid tissue.

The redistribution of cldn4 in M cells in vivo from tight junctions tothe cytoplasm in both mouse and human Peyer's Patches demonstrates thatthis transmembrane protein might participate in particle transcytosis.In support of this hypothesis, the disclosure demonstrates thatlymphotoxin/TNF treatment of the human intestinal epithelium cell lineCaco-2BBe also induced redistribution of cldn4 and that many of thebacteria were found within cldn4-positive vesicles when these cells wereallowed to endocytose fluorescently labeled bacterial particles. Theseresults implicated cldn4 as a component of M cell transcytosis. Thedisclosure also demonstrates that targeting of extracellular domains ofcldn4 mediate efficient delivery of vaccine antigens to mucosal lymphoidtissue.

The disclosure demonstrates the use of short peptides for targetingvaccine antigens to M cells. These peptides are easily incorporated intorecombinant vaccine antigens as fusion proteins, so that a singleprocess could generate both antigen and delivery “vehicle.” Adjuvantscan further be used in the delivery compositions and methods. Thedisclosure shows that Clostridium perfringens enterotoxin (Cpe), aligand for cldn4 can be used in the methods and compositions of thedisclosure (as described herein). These targeting peptides can be shownto have a direct effect on delivery of “packages” to mucosal lymphoidtissues.

The disclosure provides claudin-4 binding peptides capable ofinteracting with a claudin-4. Such peptides are typically between about8 and about 50 amino acids (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 or more amino acids).In one embodiment, the disclosure provides a substantially purifiedpeptide comprising from about 8-30 amino acids and containing a tyrosineor tryptophan separated by 3-4 amino acids followed by 1 or 2 tyrosinesand a leucine. In another embodiment, the peptide comprises about 8 to30 amino acids and comprising the amino acid sequence(Y/W)(Xaa)_(3 or 4) YYXaaL (SEQ ID NO:1) wherein Xaa is any amino acid.In yet another embodiment, the disclosure provides a peptide comprisinga sequence of between 10-30 amino acids containing a sequence of(Y/W)(Xaa)_(3 or 4) YYXaaL (SEQ ID NO:1). In one embodiment, the peptidecomprises a sequence XaaXaaXaaXaa(Y/W)(Xaa)_(3 or 4) YYXaaL (SEQ IDNO:2). In another embodiment, the peptide comprises a sequenceXaaXaa(Y/W)(Xaa)_(3 or 4)Y(Y/Xaa)(L/I)XaaXaa (SEQ ID NO:3). In specificembodiment, the peptide is selected from the group consisting of:SLDAGQYVLVMKANSSYSGNYPYSILFQKF (SEQ ID NO:4), NSSYSGNYPYSILFQKF (SEQ IDNO:5), SSYSGNYPYSIL (SEQ ID NO:6), NSSYSGNYYSIL (SEQ ID NO:7),ASNSSYSGNYSIL (SEQ ID NO:8), SPWSEPAYTLAP (SEQ ID NO:9), andAPWTEHSYYLSL (SEQ ID NO:10). In yet another embodiment, the peptidecomprises at least one D-amino acid. In a further embodiment, thepeptide binds to a claudin-4. In another aspect, the peptide comprises asequence as set forth in Table 1.

Peptides of the disclosure can be synthesized by commonly used methodssuch as those that include t-BOC or FMOC protection of alpha-aminogroups. Both methods involve stepwise synthesis in which a single aminoacid is added at each step starting from the C-terminus of the peptide(See, Coligan, et al., Current Protocols in Immunology, WileyInterscience, 1991, Unit 9). Peptides of the disclosure can also besynthesized by the well known solid phase peptide synthesis methods suchas those described by Merrifield, J. Am. Chem. Soc., 85:2149, 1962; andStewart and Young, Solid Phase Peptides Synthesis, Freeman, SanFrancisco, 1969, pp. 27-62) using a copoly(styrene-divinylbenzene)containing 0.1-1.0 mMol amines/g polymer. On completion of chemicalsynthesis, the peptides can be deprotected and cleaved from the polymerby treatment with liquid HF-10% anisole for about ¼-1 hours at 0° C.After evaporation of the reagents, the peptides are extracted from thepolymer with a 1% acetic acid solution, which is then lyophilized toyield the crude material. The peptides can be purified by suchtechniques as gel filtration on Sephadex G-15 using 5% acetic acid as asolvent. Lyophilization of appropriate fractions of the column eluateyield homogeneous peptide, which can then be characterized by standardtechniques such as amino acid analysis, thin layer chromatography, highperformance liquid chromatography, ultraviolet absorption spectroscopy,molar rotation, or measuring solubility. If desired, the peptides can bequantitated by the solid phase Edman degradation.

A polypeptide and peptide comprise a polymer in which the monomers areamino acid residues which are joined together through amide bonds. Whenthe amino acids are alpha-amino acids, either the L-optical isomer orthe D-optical isomer can be used. A polypeptide encompasses an aminoacid sequence and includes modified sequences such as glycoproteins,retro-inverso polypeptides, D-amino acid modified polypeptides, and thelike. A polypeptide includes naturally occurring proteins, as well asthose which are recombinantly or synthetically synthesized. “Fragments”are a portion of a polypeptide. The term “fragment” refers to a portionof a polypeptide which exhibits at least one useful epitope orfunctional domain. The term “functional fragment” refers to fragments ofa polypeptide that retain an activity of the polypeptide. For example, afunctional fragment of a binding peptide of the disclosure includes afragment which retains the ability to bind to a claudin familypolypeptide such as claudin 4. Biologically functional fragments, forexample, can vary in size from a polypeptide fragment as small as anepitope capable of binding an antibody molecule, to a large polypeptide.

In some embodiments, retro-inverso peptides are used. “Retro-inverso”means an amino-carboxy inversion as well as enantiomeric change in oneor more amino acids (i.e., levantory (L) to dextrorotary (D)). Apolypeptide of the disclosure encompasses, for example, amino-carboxyinversions of the amino acid sequence, amino-carboxy inversionscontaining one or more D-amino acids, and non-inverted sequencecontaining one or more D-amino acids. Retro-inverso peptidomimetics thatare stable and retain bioactivity can be devised as described byBrugidou et al. (Biochem. Biophys. Res. Comm. 214(2): 685-693, 1995) andChorev et al. (Trends Biotechnol. 13(10): 438-445, 1995). The overallstructural features of a retro-inverso polypeptide are similar to thoseof the parent L-polypeptide. The two molecules, however, are roughlymirror images because they share inherently chiral secondary structureelements. Main-chain peptidomimetics based on peptide-bond reversal andinversion of chirality represent important structural alterations forpeptides and proteins, and are highly significant for biotechnology.Antigenicity and immunogenicity can be achieved by metabolically stableantigens such as all-D-and retro-inverso-isomers of natural antigenicpeptides.

In another embodiment, the peptide is produced by recombinant DNAtechniques. For example, an oligonucleotide or polynucleotide encoding apeptide of the disclosure can be expressed from an expression vector andthe resulting peptide purified using techniques known in the art (e.g.,HPLC or other chromtography techniques). The produced pepetides may besubstantially purified and used in methods of the disclosure or they maybe substantially purified and conjugated to a second molecule ofinterest. Exemplary molecules include polypeptides or peptide (e.g.,such as a growth factor, vaccine or immunogen, antibody and the like);small molecule drugs or nano-, micro-particles. Such nano-andmicro-particles are useful in diagnostic assays, drug delivery, andtherapeutics. For example, nano-and micro-particles can be used forimaging, or heating a cancer tissue to induce cell destruction.

Suitable nanoparticles are commercially available and include hollow-andsolid-nano-spheres, -cubes, -bowls, -rods, and -porous structures. Insome embodiments, the nanostructure will comprise a noble metal (e.g.,Ag, Au, Pt) or a combination of noble metals and may further include amagnetic metal. Methods of conjugating a peptide of the disclosure to ananoparticle are known in the art and include alkanethiol linkages andthe like. The signature of a noble metal nanostructure is the localizedsurface plasmon resonance. This resonance occurs when the correctwavelength of electromagnetic energy (e.g., light) strikes a noble metalnanostructure causing the plasma of conduction electrons to oscillatecollectively. The resonance oscillation is localized near the surfaceregion of the nanostructure. Such resonance is advantageous in that thenanostructure is selectively excited at a particular photon absorption,which results in the generation of locally enhanced or amplifiedelectromagnetic fields at the nanostructure surface. The resonance fornoble metal nanostructures (e.g., in the 20-500 nm range) occurs in thevisible and IR regions of the spectrum and can be measured byUV-visible-IR extinction spectroscopy. In some nanostructures, thenanostructure can be tuned to generate a particular absorbance andemission spectra by adjusting the metallic composition and geometry.

A peptide of the disclosure useful for targeting the nanostructure to anendothelial or epithelial tissue or cell may be conjugated to thenanostructure by any number of techniques. The peptide may also belinked (or the nanostructure linked) to a second polypeptide comprisinga desired small molecule drug or polypeptide.

The nanostructures may be functionalized. The term “functionalized” ismeant to include structures with two or more layers of different metals,structures with functional groups attached thereto, structures that haveoptical properties, magnetic structures, etc. The nanostructures of canoptionally be functionalized by imprinting functional groups, such asantibodies, proteins, nucleic acids, and the like. Such nanostructuresare particularly useful for molecular diagnostics. For example, toprolong or target analyte interaction with the noble metal nanoparticlesurface, a binding agent/targeting domain is used to promote interactionof a nanostructure with a desired target. In one embodiment, thenanostructure is functionalized with a peptide of the disclosure thatinteracts with a claudin-4. In one embodiment, the functionalizednanostructure comprising a claudin-4 binding peptide is useful fortargeted delivery to an endothelial or epithelial cell or tissue. Analkanethiol, such as 1-decanethiol, can be used to form the capturelayer on the noble metal (Blanco Gomis et al., J. Anal. Chim. Acta436:173 [2001]; Yang et al., Anal. Chem. 34:1326 [1995]). Otherexemplary capture molecules include longer-chained alkanethiols,cyclohexyl mercaptan, glucosamine, boronic acid and mercapto carboxylicacids (e.g., 11-mercaptoundecanoic acid).

Alternatively, a self-assembled monolayer (SAM) is formed on thenanostructure surface to concentrate the analyte of interest near thesurface of the nanostructure. Exemplary SAMs include, but are notlimited to, 4-aminothiophenol, L-cystein, 3-mercaptopropionicacid,11-mercaptoundecanoic acid, 1-hexanethiol, 1-octanethiol, 1-DT,1-hexadecanethiol, poly-DL-lysine, 3-mercapto-1-propanesufonic acid,benzenethiol, and cyclohexylmercaptan. Typically the SAM is comprised ofstraight chain alkanethiols.

As described above, a targeting ligand (e.g., a binding peptide) caninclude a claudin-4 binding peptide bound to the surface of ananostructure such that the nanostructure interacts reversibly orirreversibly with a specific analyte. Typically, the interaction of thetargeting ligand and the analyte lasts sufficiently long for detectionof the analyte by SERS or to promote uptake and delivery of an attachedpayload.

In one embodiment, nanoparticles may be formed from compatible polymersand biomaterials such as poly(lactide-co-glycolide) (PLGA),poly(lactide) (PLA), poly ε-caprolactone, albumin, and chitosan. Incertain embodiments, the carrier particles are formed from thebiodegradable polymers PLGA or PLA. PLGA and PLA are able to control therelease of bioactive macromolecules (e.g., therapeutic agents). PLGA isa well-studied polymer for drug delivery and is FDA-approved for anumber of in vivo applications. PLGA degradation times can be variedfrom days to years by altering the type of polymer, the polymermolecular weight, or the structure of the nanospheres. Further,modifying the end groups of PLGA with acid groups (COOH) allows forgreater conjugation of peptide or protein molecules thereby allowing thenanoparticles to be targeted to specific cell types.

In some embodiments, the carrier particles are associated with atherapeutic agent (e.g., the therapeutic agent is entangled, embedded,incorporated, encapsulated, bound to the surface, or otherwiseassociated with the carrier particle). In certain embodiments, thetherapeutic agent is a drug such as a pure drug (e.g., drugs processedby crystallization or supercritical fluids), an encapsulated drug (e.g.,polymers), a surface associated drug (e.g., drugs that are adsorbed orbound to the carrier particle surface), a complexed drug (e.g., drugsthat are associated with the material used to form the carrierparticle). In a different embodiment, the carrier particles exhibitfluorescent activity or a measurable signal when exposed to light oranother external stimulus, which is useful for diagnostics, imaging andsensoring.

The carrier particles of the disclosure, in certain embodiments, do notinclude a functional group. In other aspects, however, the carrierparticles can include a functional group such as, for example, acarboxyl, sulfhydryl, hydroxyl, or amino group, or any other functionalgroup that can be used to bind a targeting moiety or moieties to thesurface of the carrier particles.

The carrier particles may be formed by suitable means known in the art,for example microspheres may be formed as described in U.S. Pat. No.7,083,572, and nanoparticles may be formed as described in U.S. Pat. No.6,632,671. It is also known in the art how to incorporate or encapsulateone or more therapeutic agents in the carrier particles for delivery.Large porous particles may be prepared using conventional techniquessuch as spray drying or emulsion solvent evaporation, or throughsupercritical fluid derived processes such as those described by Koushik& Kompella (2004) Pharm. Res. 21:524-35.

In specific embodiments, a PLGA micro-or nano-particle is linked to aclaudin-4 binding peptide to promote targeting to endothelial andepithelial tissues. The PLGA may have incorporated thereon or therein atherapeutic agent such as a vaccine or small molecule drug.

In another aspect, the disclosure provides a method of producing afusion polypeptide comprising a binding peptide domain and aheterologous molecule by growing a host cell comprising a polynucleotideencoding the fusion polypeptide under conditions that allow expressionof the polynucleotide, and recovering the fusion polypeptide. Apolynucleotide encoding a fusion polypeptide of the disclosure can beoperably linked to a promoter for expression in a prokaryotic oreukaryotic expression system. For example, such a polynucleotide can beincorporated in an expression vector. In addition, fusion polypeptidemay be generated comprising two coding domains operably linked in-framesuch that upon expression both domains retain a functional activity. Forexample, a useful fusion construct of the disclosure comprises a codingsequence for a claudin-4 binding peptide of the disclosure operablylinked (e.g., directly or via a linker) to a peptide or polypeptide ofinterest. Such a peptide or polypeptide of interest can be a growthfactor, antibody, immunogenic peptide or polypeptide molecule and thelike.

Delivery of a polynucleotide of the disclosure can be achieved byintroducing the polynucleotide into a cell using a variety of methodsknown to those of skill in the art. For example, a construct comprisingsuch a polynucleotide can be delivered into a cell using a colloidaldispersion system. Alternatively, a polynucleotide construct can beincorporated (i.e., cloned) into an appropriate vector. For purposes ofexpression, the polynucleotide encoding a fusion polypeptide of thedisclosure may be inserted into a recombinant expression vector. Theterm “recombinant expression vector” refers to a plasmid, virus, orother vehicle known in the art that has been manipulated by insertion orincorporation of a polynucleotide encoding a fusion polypeptide of thedisclosure. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes that allow phenotypicselection of the transformed cells. Vectors suitable for such useinclude, but are not limited to, the T7-based expression vector forexpression in bacteria (Rosenberg et al., Gene, 56:125, 1987), thepMSXND expression vector for expression in mammalian cells (Lee andNathans, J. Biol. Chem., 263:3521, 1988), baculovirus-derived vectorsfor expression in insect cells, cauliflower mosaic virus, CaMV, andtobacco mosaic virus, TMV, for expression in plants.

Depending on the vector utilized, any of a number of suitabletranscription and translation elements (regulatory sequences), includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, and the like may be used in the expressionvector (see, e.g., Bitter et al., Methods in Enzymology, 153:516-544,1987). These elements are well known to one of skill in the art.

The term “operably linked” or “operably associated” refers to functionallinkage between the regulatory sequence and the polynucleotide regulatedby the regulatory sequence. The operably linked regulatory sequencecontrols the expression of the product expressed by the polynucleotide.The term “operably linked” also include linking of two heterologous orhomologous domains through a linker or other moiety such that eachdomain is functional.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. (Current Protocols in Molecular Biology, Vol. 2,Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13,1988; Grant et al., “Expression and Secretion Vectors for Yeast,” inMethods in Enzymology, Eds. Wu & Grossman, Acad. Press, N.Y., Vol. 153,pp. 516-544, 1987; Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C.,Ch. 3, 1986; “Bitter, Heterologous Gene Expression in Yeast,”Methods inEnzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp.673-684, 1987; and The Molecular Biology of the Yeast Saccharomyces,Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982).A constitutive yeast promoter, such as ADH or LEU2, or an induciblepromoter, such as GAL, may be used (“Cloning in Yeast,” Ch. 3, R.Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. D M Glover,IRL Press, Wash., D.C., 1986). Alternatively, vectors may be used whichpromote integration of foreign DNA sequences into the yeast chromosome.

An expression vector can be used to transform a target cell. By“transformation” is meant a permanent genetic change induced in a cellfollowing incorporation of a polynucleotide exogenous to the cell. Wherethe cell is a mammalian cell, a permanent genetic change is generallyachieved by introduction of the polynucleotide into the genome of thecell. By “transformed cell” is meant a cell into which (or into anancestor of which) has been introduced, by means of molecular biologytechniques, a polynucleotide encoding a fusion polypeptide comprising abinding peptide linked to a heterologous polypeptide or fusogenicpolypeptide. Transformation of a host cell may be carried out byconventional techniques as are known to those skilled in the art. Wherethe host is prokaryotic, such as E. coli, competent cells which arecapable of polynucleotide uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method by procedures well known in the art. Alternatively, MgCl₂ or RbClcan be used. Transformation can also be performed after forming aprotoplast of the host cell or by electroporation.

A fusion polypeptide of the disclosure can be produced by expression ofpolynucleotide encoding a fusion polypeptide in prokaryotes. Theseinclude, but are not limited to, microorganisms, such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA, or cosmidDNA expression vectors encoding a fusion polypeptide of the disclosure.The constructs can be expressed in E. coli in large scale for in vitroassays. Purification from bacteria is simplified when the sequencesinclude tags for one-step purification by nickel-chelate chromatography.Thus, a polynucleotide encoding a fusion polypeptide can also comprise atag to simplify isolation of the fusion polypeptide. For example, apolyhistidine tag of, e.g., six histidine residues, can be incorporatedat the amino terminal end of the fusion polypeptide. The polyhistidinetag allows convenient isolation of the protein in a single step bynickel-chelate chromatography. A fusion polypeptide of the disclosurecan also be engineered to contain a cleavage site to aid in proteinrecovery or other linker moiety separating a binding peptide from aheterologous molecule (e.g., an immunogenic/vaccine polypeptide orpeptide). Typically a linker will be a peptide linker moiety. The lengthof the linker moiety is chosen to optimize the biological activity ofthe polypeptide comprising binding peptide domain and a heterologousmolecule and can be determined empirically without undueexperimentation. The linker moiety should be long enough and flexibleenough to allow a binding peptide polypeptide to freely interact. Alinker moiety is a peptide between about one and 30 amino acid residuesin length, typically between about two and 15 amino acid residues.Examples of linker moieties are -Gly-Gly-, GGGGS (SEQ ID NO:13) one ormore times, GKSSGSGSESKS (SEQ ID NO:14), GSTSGSGKSSEGKG (SEQ ID NO:15),GSTSGSGKSSEGSGSTKG (SEQ ID NO:16), GSTSGSGKPGSGEGSTKG (SEQ ID NO:17), orEGKSSGSGSESKEF (SEQ ID NO:18). Linking moieties are described, forexample, in Huston et al., Proc. Nat'l Acad. Sci 85:5879, 1988; Whitlowet al., Protein Engineering 6:989, 1993; and Newton et al., Biochemistry35:545, 1996. Other suitable peptide linkers are those described in U.S.Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated byreference. A DNA sequence encoding a desired peptide linker can beinserted between, and in the same reading frame as, a polynucleotideencoding a claudin-4 binding peptide or fragment thereof followed by aheterologous polypeptide, using any suitable conventional technique. Forexample, a chemically synthesized oligonucleotide encoding the linkercan be ligated between two coding polynucleotides. In particularembodiments, a fusion polypeptide comprises from two to four separatedomains (e.g., a binding peptide domain and a heterologous polypeptidedomain) are separated by peptide linkers.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical procedures,such as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransfected with a polynucleotide encoding the binding peptide-fusionpolypeptide of the disclosure, and a second polynucleotide moleculeencoding a selectable phenotype, such as the herpes simplex thymidinekinase or cytosine deaminase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982).

Eukaryotic systems, and typically mammalian expression systems, allowfor proper post-translational modifications of expressed mammalianproteins to occur. Eukaryotic cell s that possess the cellular machineryfor proper processing of the primary transcript, glycosylation,phosphorylation, and advantageously secretion of the gene product can beused as host cells for the expression of the binding peptide-fusionpolypeptide of the disclosure. Such host cell lines may include, but arenot limited to, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, andWI38.

For long-term, high-yield production of recombinant proteins, stableexpression is useful. Rather than using expression vectors that containviral origins of replication, host cells can be transformed with thecDNA encoding a fusion polypeptide of the disclosure controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, and thelike), and a selectable marker. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci that,in turn, can be cloned and expanded into cell lines. For example,following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. A number of selection systems may be used,including, but not limited to, the herpes simplex virus thymidine kinase(Wigler et al., Cell, 11:223, 1977), hypoxanthine-guaninephosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.USA, 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy et al.,Cell, 22:817, 1980) genes can be employed in tk-, hgprt-or aprt-cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA, 77:3567, 1980; O'Hare et al., Proc.Natl. Acad. Sci. USA, 8:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072,1981; neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol., 150:1, 1981); and hygro, whichconfers resistance to hygromycin genes (Santerre et al., Gene, 30:147,1984). Additional selectable genes have been described, namely trpB,which allows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA, 85:8047, 1988); and ODC (ornithinedecarboxylase), which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DEMO (McConlogue L., In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory, ed., 1987).

Techniques for the isolation and purification of either microbially oreukaryotically expressed binding peptide-fusion polypeptides of thedisclosure may be by any conventional means, such as, for example,preparative chromatographic separations and immunological separations,such as those involving the use of monoclonal or polyclonal antibodiesor antigen.

Small cyclic peptides may generally be used to specifically modulateadhesion of cancer and/or other cell types by topical administration orby systemic administration, with or without linking a targeting agent tothe peptide, as discussed below.

A pharmaceutical composition according to the disclosure can be preparedto include a polypeptide of the disclosure, into a form suitable foradministration to a subject using carriers, excipients, and additives orauxiliaries. Frequently used carriers or auxiliaries include magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk protein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol, and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents, and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487(1975), and The National Formulary XIV., 14 th ed., Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's, The PharmacologicalBasis for Therapeutics (7th ed.).

The pharmaceutical compositions according to the disclosure may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the disclosurenecessary to prevent, to cure, or at least partially arrest the symptomsof tissue damage. Amounts effective for this use will, of course, dependon the severity of the disease and the weight and general state of thepatient. Typically, dosages used in vitro may provide useful guidance inthe amounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of particular disorders. Various considerationsare described, e.g., in Langer, Science, 249: 1527, (1990); Gilman etal. (eds.) (1990), each of which is herein incorporated by reference.

As used herein, “administering a therapeutically effective amount” isintended to include methods of giving or applying a pharmaceuticalcomposition of the disclosure to a subject that allow the composition toperform its intended therapeutic function. The therapeutically effectiveamounts will vary according to factors, such as the degree of infectionin a subject, the age, sex, and weight of the individual. Dosage regimacan be adjusted to provide the optimum therapeutic response. Forexample, several divided doses can be administered daily or the dose canbe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The pharmaceutical composition can be administered in a convenientmanner, such as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids, and othernatural conditions that may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size, in the case ofdispersion, and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols, such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepharmaceutical composition in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the pharmaceutical composition into a sterilevehicle that contains a basic dispersion medium and the required otheringredients from those enumerated above.

The pharmaceutical composition can be orally administered, for example,with an inert diluent or an assimilable edible carrier. Thepharmaceutical composition and other ingredients can also be enclosed ina hard or soft-shell gelatin capsule, compressed into tablets, orincorporated directly into the individual's diet. For oral therapeuticadministration, the pharmaceutical composition can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationscan, of course, be varied and can conveniently be between about 5% toabout 80% of the weight of the unit.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: a binder, such as gum gragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agent,such as corn starch, potato starch, alginic acid, and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin, or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar, or both.A syrup or elixir can contain the agent, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye, and flavoring, suchas cherry or orange flavor. Of course, any material used in preparingany dosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the pharmaceuticalcomposition can be incorporated into sustained-release preparations andformulations.

Thus, a “pharmaceutically acceptable carrier” is intended to includesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the pharmaceutical composition, use thereof in thetherapeutic compositions and methods of treatment is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form” as used herein, refers to physically discrete unitssuited as unitary dosages for the individual to be treated; each unitcontaining a predetermined quantity of pharmaceutical composition iscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the noveldosage unit forms of the disclosure are dictated by and directlydependent on: (a) the unique characteristics of the pharmaceuticalcomposition and the particular therapeutic effect to be achieve, and (b)the limitations inherent in the art of compounding such anpharmaceutical composition for the treatment of a pathogenic infectionin a subject.

The principal pharmaceutical composition is compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Materials. Peptides used in this study were synthesized by Abgent (SanDiego, Calif.) and AnaSpec (San Jose, Calif.). Peptides were synthesizedby solid-phase synthesis procedure, purified by reverse phase HPLCto >98% purity. The sequences of peptides used in the disclosure aresummarized in Table 1. For immobilization to Streptavidin (SA) sensorchip (Biacore, GE), peptides were biotinylated at N-or C-terminus withGS (GGGGS) (SEQ ID NO:13) or PEG linker to increase the accessibility ofpeptide. Peptides were dissolved in a small amount of solvent (e.g.,DMSO at 5-10% final concentration) and brought up to specificconcentrations by H₂O; the pH was adjusted to neutral by phosphatebuffer. Primers for subcloning were synthesized by IDT.

TABLE 1 Peptides and Claudin-4 Mimics Used in This Study PeptideSequence Structure Cpe30 SLDAGQYVLVMKANSSYSGNYPYSILFQKFBiotin(B)-PEG10-Cpe30 Cpe17              NSSYSGNYPYSILFQKF B-PEG6-Cpe17Cpe30 MT1               SSYSGNYPYSIL B-PEG8-Cpe30MT1 Cpe30 MT2             NSSYSGNYYSIL B-PEG8-Cpe30MT2 Cpe30 MT3           ASNSSYSGNYSIL B-PEG8-Cpe30MT3 CC4P-5              SPWSEPAYTLAP CC4P-5-PEG8-B CC4P-13              APWTEHSYYLSL CC4P-13-PEG8-B CC4GP-1              APWHLSSQYSRT B-PEG8-CC4GP-1 CC4GP-1Y              APYHLSSQYSRT B-PEG8-CC4GP-1Y CC4P-2 VTHKTCPPACWPCC4P-2-PEG8-B Cldn4 mimics Ecl2 (138)AHNVIRDFYNPMVASGQKREMGASL(160)B-2 GS(GGGGS)-Ecl2 Tethered Ecl2 AHNVIRDFYNPMVASGQKREMGASLB-PEG4-Ecl2-PEG4-B Lariat Ecl2 AHNVIRDFYNPMVASGQKREMGASLB-PEG10-Cys-Ecl2-Cys GST-Ecl2 GST-Aa 138-160

   -FL Aa 1-210

   -TM3 Ecl2.TM4 Aa 117-181

   -TM3 Ecl2.CT Aa 117-210

   -Ecl2.CT Aa 138-210

Note: Peptides were synthesized as a biotinylated form to be conjugatedinto streptavidin sensor chip. PGE or GS linker was added to increasethe accessibility of peptide. Claudin-4 deletion mutants were expressedas a GST recombinant protein to improve the solubility of this membraneprotein.

Screening Cldn4 binding peptides by phage display library. A random12-mer peptide phage display library (Cat# E8110S, NEB, USA) was usedfor screening. The library size is about 2.7×10⁹ transformants. Toselect phage against transmembrane Cldn4 extracellular domains, CHOcells transfected with full length Cldn4 (CHO-Cldn4) were employed forphage library selection. The screening was performed according to themanufacture's instruction with some modifications. Four rounds ofpanning were done, where the 1st round was selected by CHO-Cldn4 cells(“positive selection”). The 2nd round used CHO control cells (“negativeselection”) to remove phage specific for CHO determinants. This second,negatively selected library was followed by a 3^(rd) positive selectionround with CHO-Cldn4 cells, and an additional 4^(th) negative selectionround with CHO control cells. Phage clones were then isolated forsequencing of display peptide sequences, and abundant or repeatedsequences were selected as candidate clones. Two positive bindingpeptides (CC4P-13, CC4P-5) (Table 1) were selected after 4 rounds ofscreening. The peptides were synthesized for SPR assay.

Protein expression and purification. Claudin-4: Mouse claudin-4 (cldn4,NM_(—)00903) was subcloned into pGEX4T-2 by BamHI(5′) and XhoI (3′) byPCR high fidelity DNA polymerase Pfu (Stratagene, USA). The primers foreach deletion mutant were: full length clnd4 by F1 (forward primer 1):5′-GGATCCGCGATGGCGTCTATGGGAC-3′ (SEQ ID NO:19); R1 (reverse primer 1):5′-CTCGAGTTACACATAGTTGCTGGCGGGG-3′(SEQ ID NO:20); Ecl2 by F2:5′-GGATCCATCATGATCACCGCCGGAG-3′ (SEQ ID NO:21); R2:5′-CTCGAGTCAGAGGAGGCCTCCTCC-3′ (SEQ ID NO:22); TM3.Ecl2.CT by primer F2and R1; and Ecl2.CT by F3: 5′-GGATCCTGGACCGCTCACAACG-3′ (SEQ ID NO:23)and primer R1. The underlined sequences were BamHI and XhoI restrictionsites. The constructs were confirmed by DNA sequencing.GST-Cldn4/pGEX4T-2 construct was transformed into E. coli (BL21, pLysS)for protein expression. The soluble protein was purified byGlutathione-agarose affinity chromatography (Pierce, USA), and theco-purified GST protein was separated by gel filtration chromatographyon FPLC with Superdex 200 column. For use as the analyte in Biacoreassay, GST-cldn4 was balanced to HBS-EP buffer by Microcon (Millipore)centrifugation.

Hemagglutinin (HA): HA from influenza A virus (A/Puerto Rico/8/34/MountSinai, AF389118) was used to express recombinant HA protein. It wassubcloned into pENTR3C vector via BamHI (5′) and EcoRV (3′) by PCRprocedure, and recombined into BaculoDirect Linear DNA (“BaculoDirect™Baculovirus expression system”, Invitrogen, USA). The resultantexpression virus was used to express protein in insect cell (Sf9). TheC-terminal 37 amino acids of HA containing the transmembrane domain wereremoved to enable secretion of soluble protein, and a trimerizationsequence (ts, from Fibritin-C) was inserted to facilitate efficienttrimerization of HA. Cpe30 (the c-terminal 30 amino acids of theClostridium perfringens enterotoxin) was introduced to the C-terminuswith upstream GS linkers (FIG. 6). This recombinant HA (HA-cpe30) wasC-terminally His tagged, and purified by HisPur Cobalt (Pierce, USA)affinity chromatography.

Measurement of binding kinetics by SPR. The BiacoreX100 system (Biacore,GE) was used in this experiment. SA (streptavidin) sensor chips wereused to immobilize the biotinylated peptides. HBS-EP buffer (10 mMHEPES, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH7.4) was used asthe binding buffer. General procedures were according to manufacturer'sinstructions. Biotinylated peptide (e.g., Cpe30) was immobilized to theSA chip after first conditioning the chip surface with 50 mM NaOH/1MNaCl. The immobilized amount of the ligand was ˜500 RU (response units)to ensure efficient binding. The control channel was treated in the sameway as assay channel but without peptide immobilized. The analyte was aGST-Cldn4 protein comprised of GST fused at the C-terminal end to afragment of Cldn4 from the second extracellular domain (Ecl2) throughthe C-terminus of Cldn4. The titration was measured with purifiedGST-Cldn4 from 0.2 uM to 2 uM in HBS-EP. GST protein only was employedas the control analyte. The binding was carried out at 25 C with flowrate at 30 ul/min, and data were collected for 2 min of association and3 min of dissociation.

In the other assay, an HA conformation sensitive antibody (H36) wasimmobilized to CM5 sensor chip by amine-coupling reaction, thenrecombinant HA protein was indirectly captured as the ligand to interactwith GST-Cldn4.R4 analyte in HBS-EP buffer as above.

To analyze the data, the assay channel was subtracted by the controlchannel in order to eliminate nonspecific interaction. Multiplesensorgrams from different concentrations of analyte were overlaid andaligned, and kinetic constants were calculated by BIAevaluation 3.1software with nonlinear fitting, the 1:1 (langmuir) binding model wasused, where K_(D)=kd/ka.

Measurement of peptide binding to GST-Cldn4 by co-purification. In vitrobinding assays were carried out with biotinylated peptides (lug) andpurified GST-Cldn4.R4 (5 ug) in HBS-EP. The binding was incubated for 1hr at room temperature, followed by purification of the complex byimmobilized Neutravidin™ affinity chromatography (Pierce, USA). Theresin was washed 3 times with HBS-EP, and the bound protein was analyzedby SDS-PAGE followed by western blotting with Cldn4 antibody(Invitrogen, USA).

Claudin-4 Ecl2 in the context of transmembrane domains provides theconformation for Cpe30 binding. Claudin-4 was initially cloned as theCpe receptor (CPE-R); consequently, Cpe, as a naturally-occurringligand, has proven to be a useful molecular probe to study the functionof Cldn-4. To measure the binding affinity, various methods wereutilized. When ¹²⁵I-C-Cpe comprising amino acids 184-319 of SEQ IDNO:24:

mlsnnlnpmv fenakevfli sedlktpini tnsnsnlsdg lyvidkgdgw ilgepsvvssqilnpnetgt fsqsltkske vsinvnfsvg ftsefiqasv eygfgitige qntiersysttagpneyvyy kvyatyrkyq airishgnis ddgsiykltg iwlsktsads lgnidqgslietgercvltv pstdiekeil dlaaaterln ltdalnsnpa gnlydwrssn sypwtqklnlhltltatgqk yrilaskivd fniysnnfnn lvkleqslgd gvkdhyvdis ldagqyvlvmkanssysgny pysilfqkfwere used to interact with claudins over-expressed in mouse L cells, itwas found that C-Cpe does not bind to Cldn-1 and Cldn-2, whereas Cldn-3and Cldn-4 show specific binding with calculated affinity (Ka fromScatchard plot) of 8.4×10⁷ M⁻¹ and 1.1×10⁸ M⁻¹ respectively. In anotherstudy, a far-western approach was employed to measure the binding ofpurified C-Cpe to GST-Cldn3 Ecl2 protein separated by SDS-PAGE andblotted onto NC membrane; here, the Ka was calculated to be 1.0×10⁸ M⁻¹.Claudin-6, 7, 8 and 14 were also tested for C-Cpe binding by thismethod, with affinities estimated from submicro-to micromolar levels.

These above methods only measured the binding affinity at the end-point,and binding kinetics were not determined. Since binding on-and off-ratesmay be important parameters in the behavior of the peptides in vivo, anovel SPR method was developed used as described herein. The firststrategy was to directly use Ecl2 peptide bound to an SA chip tointeract with binding peptides. The Ecl2 sequence alone was synthesizedas a biotinylated peptide (Table 1), immobilized to SA chip as theligand, then tested against the free Cpe30 peptide as the analyte. Nospecific binding was observed from this assay (FIG. 1, upper panel). Inan attempt to better model the conformation of the cldn-4 extracellulardomain, the Ecl2 peptide was synthesized with biotin moieties at bothN-and C-termini. One rationale was that such a loop would potentially betethered to an SA chip by binding to a single tetravalent streptavidinmolecule; this could present Ecl2 in a more physiological “loop”conformation. However, the result still failed to bind to Cpe30 (FIG. 1middle panel). Another attempt at mimicking the natural Ecl2 loop wasdone by synthesis of Ecl2 as a cyclic peptide with a disulfide bondbetween two cysteines flanking the ends; this loop also failed to bind(FIG. 1 lower panel). In these attempts, the constraints on the Ecl2loop may have failed to permit proper folding of the peptide.

The results above indicated that the isolated Cldn-4 Ecl2 alone is notsufficient for binding to Cpe30. It is likely that when Cldn-4 is in thecell membrane in vivo, the upstream and downstream transmembrane domains(TM) provide additional structural contexts to maintain Ecl2conformation; these TMs may therefore be helpful in establishing thecorrect Ecl2 conformation in vitro. Thus, studies using Ecl2 in thecontext of other cldn-4 domains as shown in FIG. 2A was performed. Thesmall molecular weight of Cldn-4 with 4 TMs and a strong tendency ofoligomerization pose a difficult solubility problem for this protein. Toenhance the solubility of these constructs, GST was fused to theN-terminus; this was helpful, though there was still significant proteinin the inclusion body when expressed in BL21 (pLysS) E. coli strain.Soluble protein was purified by Glutathione-agarose affinitychromatography with further separation by gel-filtration. Theserecombinant proteins were then found to be at a purity sufficient forSPR assays (FIG. 2B).

Assays were then performed using B-Cpe30 as the ligand bound to an SAchip, and the GST-Cldn4 recombinant proteins were used as the analytes.The higher molecular weight of these analytes was also beneficial inincreasing the sensitivity of the SPR assay. When these GST cldn-4fusion proteins were tested against Cpe30, high affinity specificbinding was observed. The assay channel showed a typical association anddissociation response by comparison to the control channel, which onlyreflected fluctuations in analyte concentration. The subtractedsensorgrams at a medium concentration of each analyte were overlaid andshown in FIG. 2C. The disclosure demonstrates that all of the Cldn-4variants shown exhibited binding activity, suggesting that Ecl2 in thecontext of neighboring cldn-4 domains displayed better conformation thanas a separated peptide. Sensorgrams from all fusion proteins behavednormally except for the fusion containing the full length Cldn-4. Thisprotein showed a slight decrease in the late stages of association,raising the possibility of a two-phase interaction from interactionswith Ecl1 or other parts of Cldn-4 sequence.

TM3.Ecl2.TM4 expressed a consistent binding to Cpe30, indicating thatthe C-terminal domain of Cldn-4 was not required for interaction withligand, even though this domain is thought to be involved in thesignaling functions of Cldn-4 in vivo. By removing the TM3 and addingthe C-terminal tail (the R4 construct), the fusion protein now displayedoptimal binding activity to Cpe30. Presumably, the GST moiety at theN-terminus could serve as an anchor similar to TM3. Therefore, the R4construct was chosen as the best mimic of Ecl2 for the rest of thisstudy. It was interesting to note that GST-Ecl2 without any TM domainalso exhibited a detectable binding activity, albeit at much loweraffinity. This may in part be due to GST dimerization, helping Ecl2 forma partial “loop” conformation.

Claudin-4 binding peptides exhibit different kinetics but share a commonbinding motif. With the establishment of this SPR method, the kineticsof binding of Cpe30 and Cpe17 to cldn-4 Ecl2 was tested. Deletionexperiments have demonstrated that the last 30 amino acids of Cpe wereresponsible for the binding to Cldn-4. Loss of the 17 terminal aminoacids also caused the loss of binding, raising the possibility that thisregion contained the minimal binding domain. Therefore, B-Cpe30 andCpe17 immobilized to an SA chip as the ligand were tested, and a seriesof concentrations of GST-Cldn4.Ecl2 (R4) as the analyte was applied tomeasure binding kinetics. The results demonstrated a strong specificbinding; the subtracted sensorgrams were overlaid as shown in FIG. 3.GST protein as a control did not bind Cpe30, confirming that thespecific binding was from the Cldn-4 Ecl2. The 50 nM analyteconcentration was repeated twice to ensure the reproducibility of theassay. The calculated equilibrium affinities (K_(D)) for Cpe30 and Cpe17were 2.56 nM and 1.57 nM respectively, which was in the typical range ofreceptor-ligand binding. Kinetics analysis showed the on-rates weresimilar for Cpe30 and Cpe17, but Cpe17 had a slightly slower off-rate,giving it a higher calculated equilibrium affinity.

This SPR method also provided a useful tool to evaluate candidatepeptides selected by phage display. To generate new short peptides withCldn-4 binding activity, a phage display approach was used. A largelibrary of random 12-mer display peptides allowed us to select forpeptides able to bind cldn-4 under physiological conditions in vitro.Claudin-4 over-expressed in CHO cells was used as the bait for selectionusing alternating rounds of positive selection on transfected CHO cells,and negative selection against non-transfected cells. Starting after thethird round, phage clones were sequenced, and sequences showingenrichment with successive rounds of selection were considered candidatebinding clones. Peptides encoded by these sequences were thensynthesized (with a peptide or PEG spacer, ending with a biotin forbinding to the SA chip) and tested for specific binding to cldn-4. Threeabundant peptides (CC4P-13, -5 and -2) were identified after 4 rounds ofscreening (Table 1). CC4P-13 was biotinylated at the C-terminus toreproduce the orientation of the peptide when displayed on the surfaceof the phage particle. By contrast, CC4P-13R was biotinylated atN-terminus; this orientation is similar to that in the recombinant HAfusion protein described below. The assays were performed as for Cpe30,and the sensorgrams shown in FIG. 3 demonstrated a clear specificbinding of CC4P-13 and CC4P-5 peptides to Ecl2. The K_(D) was measuredat the nanomolar level, with CC4P-13R showing the slowest off-rate.CC4P-2 showed no binding to Ecl2 by this SPR assay. This could be due toseveral factors; this clone might be specific for CHO determinants andsimply survived negative selection, it could be specific for the cldn-4Ecl1 domain, or other unknown factors enabled its enrichment.

The SPR assay proved to be helpful in determining the specific bindingand the detailed kinetics of several cldn-4-binding peptides.Interestingly, the orientation of biotinylation did not affect thebinding of CC4P-13 to Ecl2, reinforcing the notion that this peptide wasa true cldn-4 ligand. Such adaptable peptides could have utility in avariety of other contexts.

The disclosure shows that the binding activity of Cpe30 was abolishedupon removal of the last five amino acids. Since function 12-merpeptides were identified, the possibility of a common binding motif forCldn4 Ecl2 was analyzed. Based on the CC4P-13 and CC4P-5 similarities toCpe30 and Cpe17, the basic Cpe30 sequence was reduced to a 12-mer. Thiswas done by removing the last 4 amino acids and the N-terminal 13 aminoacids not included in Cpe17, then the spacing between the Y306, Y310 andY312 was modified with minimal changes in amino acid composition (Table1). Three candidate mutant 12-mers (MT1 through MT3) were tested usingthe same SPR assay as in FIG. 3. MT2 was found to possess bindingactivity to Ecl2 with high affinity (FIG. 4), although MT1 and MT2showed no specific binding.

Comparing the sequences among these positive binding peptides revealedan interesting pattern. As shown in FIG. 5, all of the peptidescontained tyrosine or tryptophan at the position corresponding to theY306 in Cpe, and after a 3-4 amino acids spacer there were 1 or 2tyrosines followed by a leucine. This pattern constituted an apparentEcl2 binding motif; it is likely that the first Y or W is the priming ordocking site and the remaining Y and L residues participate in thechemical bonding of the contact.

Claudin-4 binding peptide retains binding activity in recombinantprotein. Peptides with the ability to bind to cldn-4 in vivo could havemany useful applications, including targeting delivery vehicles totumors over-expressing cldn-4, or targeting vaccine antigens to mucosalM cells. For this purpose, it would be important to know whether thepeptide can still function in other contexts, such as part of arecombinant fusion protein. Accordingly, tests were performed todetermine whether the attachment of a targeting peptide to thec-terminal end of a recombinant influenza hemagglutinin (HA) would stillretain targeting specificity.

To test this question, Cpe30 was integrated into the C-terminus of HA.The recombinant fusion protein (HA-ts-Cpe30) was produced by an insectexpression system and purified for the SPR assay. The recombinantprotein was captured on a CM5 chip using an anti-HA antibody (H36),which is specific for a conformational determinant on the globular headof the HA trimer. In this orientation, the HA trimer is presented withthe c-terminal tail and Cpe30 domain displayed outwards for interactionwith analyte. The binding to GST-Cldn4.R4 was tested as in FIG. 3. TheHA fusion protein with Cpe30 exhibited strong specific binding to Ecl2,though the association seemed to be a 2-phase reaction and the on-ratewas slower than free Cpe30 peptide. This is possibly due to thedecreased surface accessibility caused by the c-terminal His tag. Cpe30in HA protein could also be affected by flanking sequences to someextent, even though it was predicted to be exposed on the proteinsurface (Protean software (Lasergene)). When HA protein without Cpe30was used as the ligand, no specific interaction was detectable. Theaddition of a cldn-4-binding peptide to a recombinant globular proteinstill showed binding activity even when in a heterologous context. Thisprinciple could have application in a variety of useful situations.

The active uptake of fluorescent particles was examined when coated witheither a control peptide or with the cldn4 targeting peptide Cpe30.Beads were instilled into the nasal passage of BALB/c mice, and tenminutes later the NALT was removed for histological examination of beaduptake into the NALT follicle. The Cpe30-coated beads were taken up atsignificantly higher levels relative to control beads coated with ascrambled sequence from CC4p-13 (FIGS. 7 and 8). As another type ofcontrol, targeted uptake was examined in CD137/4-1BB knockout mice,which demonstrate defective M cell function; here the uptake of Cpe30coated beads was only at background levels, showing the requirement foractive uptake of the targeted beads. Differences between Cpe30 coatedbead uptake in BALB/c and either control peptide-coated beads or CD137knockout mice were highly significant (*p<0.0001).

M cell targeting of mucosal vaccines: Tissue specific titers and isotyperesponses. As noted above, an expression vector was developed that couldincorporate the target vaccine antigen, fused with functional subunits,including spacer peptides, trimerization peptide, and M cell-targetingpeptide. The first target organism was influenza. A recombinant versionof the influenza hemagglutinin (HA) was produced, as neutralizingantibodies against HA would be best at blocking viral entry. A truncatedform of HA lacking the transmembrane and cytoplasmic domains wasproduced. Such a recombinant protein forms trimers in solution, but toencourage effective trimer formation, a trimerization peptide fromFibritin-C flanked by peptide spacers was added at the C-terminal end.The M cell-targeting Cpe30 peptide, with a His tag at the end to assistin purification, was then placed at C-terminal to the trimerizationpeptide. The map of the expression construct is shown in FIG. 9. Thenthe recombinant protein was produced in baculovirus-insect cellcultures, the presence of the trimerization peptide helped stabilize HAtrimers even when analyzed by non-denaturing gels (FIG. 10). Properconformation of the protein trimers was confirmed by experimentsdemonstrating that the trimeric protein bound to aconformation-dependent monoclonal antibody against HA (H36). Thisprotein proved to have cldn4-binding with similar affinity as the freeCpe30, demonstrating that the targeting peptide retained function evenin the context of a fusion protein. For immunization controls, HAwithout the targeting Cpe30 peptide was generated.

Various versions of the targeted HA vaccine were tested in mucosalimmunizations of mice. BALB/c mice (5 mice per group) were immunizedwith three weekly intranasal doses of 2 μg HA-ts-Cpe30 or control HAwith cholera toxin (1 μg) in the first dose only. On the fourth week,both groups developed similar serum IgG titers against HA, as revealedby the ELISA titration curves. In contrast to IgG titers, the targetedHA group (HA-Cpe) developed higher IgA anti-HA titers in the intestinalcontent (IC) than the control group (HA) (FIG. 11). In a second study,HA chemically conjugated with Cpe30 or control peptide (a scrambledpeptide sequence) was used for intranasal immunization at 20 μg perdose, in combination with 5 μg flagellin per dose as an adjuvant. Ahigher antigen dose was required with the chemical conjugation, perhapsbecause the conjugation may have reduced the overall antigenicity of therecombinant HA. In this study, the targeted HA (HA-cpe) induced higherserum IgG and intestinal content IgA titers against HA than thenon-targeted HA, with significantly greater intestinal IgA/IgG ratio inthe targeted group than in control mice (FIG. 12). In an additionalstudy, the purified flagellin was replaced with an HA-flagellinrecombinant fusion protein, similar to the HA-ts-Cpe30-HT construct, butwith the Cpe30 sequence replaced by a full length sequence of flagellincloned from Salmonella typhimurium. When mixed with HA-Cpe30, thiscombination induced the highest intestinal and serum IgA titers of all(FIG. 13). Collectively, these three examples indicate that thetargeting of the vaccine antigen to mucosal lymphoid tissues (along witha mucosal adjuvant) specifically boosts mucosal IgA responses.

Antibody responses: changing titers versus overall affinity. The immuneresponse of immunized animals is expected to decay over time in theabsence of booster immunizations or infection. The ability of a decayingantibody response to provide persistent protective immunity will dependin large part on the persistence of neutralizing titers in the mostimportant tissues (e.g., mucosal tissues) and the affinity of theantibodies present. The affinity of Abs may have important implicationson viral infection, as lower affinity Abs may enhance viral infection ofmyeloid cells that express Fc receptors via the ADE phenomenon.

In this context, both ELISA and the Biacore/Surface Plasmon Resonance(SPR) method were used to assess whether the method of immunization mayinfluence not only the antibody titer, but also the affinity of theantibodies for the target antigen. SPR can be used to measure not onlyequilibrium affinity but also on-and off-rates of protein binding. Thesemeasurements are best performed with highly defined purified proteins,but they can also be used for qualitative studies on polyclonal antibodybinding to target antigen. In studies, antibodies in serum andintestinal contents were compared from mice immunized with HA versus theM cell-targeted HA-CPE30 vaccine (FIG. 14). Precise calculations ofantibody affinity are not possible for polyclonal responses, as theexact concentration of the HA-binding antibodies (relative to total Ig)cannot be measured. However, a difference in the binding of antibodiesfrom the HA-CPE30 immunization versus HA alone or preimmune antibodieswas observed. Specifically, the higher shift in resonance units inducedby serum from the HA-CPE30 immunized mouse suggests a higher affinityresponse than seen in the mouse immunized by HA.

The disclosure demonstrates that the vaccination studies provideproof-of-principle data for the ability of an M cell-targeted vaccine tospecifically induce high intestinal IgA titers against a vaccineantigen. The intranasal or oral route of immunization allows the Mcell-targeted fusion protein to be potentially applicable as aneedle-free vaccine that could be used in human populations. Althoughhumans have tonsils instead of NALT, studies have confirmed that cldn4is highly expressed in tonsil crypt epithelium, where the concentrationof tonsil M cells is highest. In the context of the disclosure, this newvaccine delivery technology could be used as an effective means ofinducing high intestinal IgA titers against respiratory and intestinalinfectious organism antigens.

The disclosure provides a method for PLGA nanoparticle production thatprovides useful features for delivery to mucosal cells. First, theparticles are in a narrow size range around 300 nanometers, which are anoptimal size range for mucosal M cell uptake. Second, the protocol canbe adapted to incorporate nearly any new protein, and the particles showa two phase release profile with a rapid phase releasing up to half ofthe protein over a few days, and a slow phase releasing the rest of theprotein over three months. Third, the particles display most of theprotein on the surface of the particles (probably accounting for thefast release phase). This presentation of the protein on the surface isquite different from particles using polymers such as PLA:PEG blockco-polymers, where the protein is mainly encapsulated within the matrix.The important consequence of this distribution is that the targetingpeptide on the HA-CPE protein is accessible and thus can be used for Mcell targeting of the particles both in NALT and Peyer's patches.Studies in the literature suggest that PLGA might help protect theprotein from digestion in the intestine.

Manufacture of vaccine HA antigen, and new HA construct with enhancedbinding. The disclosure has demonstrated that CPE peptides and fragmentsbind to Claudin 4. Claudin 4 is a target receptor on mucosal M cells,and so a recombinant fusion proteins with the influenza hemagglutinin(HA) attached to a CPE30 peptide was developed to measure whether thefusion construct was a suitable vaccine.

Method for producing PLGA nanoparticles incorporating proteins fortargeting to mucosal M cells. The objective is to develop a method forpreparation of biodegradable nanoparticles loaded with recombinantproteins with targeting peptides for optimal M cell uptake.

Materials for nanoparticle preparation: PLGApoly(DL-lactide-co-glycolide (85:15 PLGA, MW 50,000-75,000)Sigma-Aldrich Catalog #430471-5G; Poly(vinyl alcohol) (PVA, MW30,000-70,000, 87%-90% hydrolyzed) Sigma-Aldrich #P8136-250G;4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid (HEPES, 1M),Invitrogen #15630-080; Phosphate Buffered Saline (PBS, 1X) Invitrogen#10010; Sodium Dodecyl Sulfate solution (10% SDS), Invitrogen#24730-020; F-12 Kaighn's medium Invitrogen #21127; geneticin Invitrogen#10131-035; Methylene Chloride optima®, Fisher #D151-1; PBS (10× readyconcentrate pouches), Fisher #BP665-1; HEPES (powder fine whitecrystals) Fisher #BP310-500; sodium hydroxide (certified A.S.C) Fisher#S318-500; Rhodamine 6G Sigma-Aldrich #83697; 16% paraformaldehydeElectron Microscopy Services #15710; Prolong Gold antifade reagent withDAPI Molecular Probes #P36935; Pierce BCA™ Protein Assay Kit Fisher#23227 Branson® sonifier 450.

Procedure: Nanoparticle Preparation. PLGA nanoparticles containingtargeting (HA-HT-CPE30) and non-targeting (HA-HT) peptides were preparedfrom 85:15 PLGA using solvent evaporation/double emulsion (also known aswater-in oil-in water, w/o/w) method.

Preparation of stock solutions. 4% PLGA polymer solution was prepared byadding 0.18 g of PLGA into 4.5 mL of metheylene chloride in a glassbeaker and stirring until dissolved. 2% PVA solution was prepared bydissolving 0.6 g of PVA in 30 ml 10 mM HEPES and adjusting the pH to 7.5with NaOH. Protein solutions: HA-HT-CPE30 or HA-HT protein in HEPESbuffer with 3.0-4.5 mg/ml concentration. For labeling experiments; a 40mg/ml Rhodamine 6 G (R6G) solution was prepared by dissolving 1 mg ofR6G in 25 μl of metheylene chloride.

Preparation of first emulsion: The reagents listed in the table wereadded to a 18×150 mm disposable glass tube in the order listed.

4% PLGA solution 4.25 ml Protein 0.5 ml of HA•HT•CPE30 or HA•HT 2% PVAStabilizer 0.25 mlFor labeled nanoparticles, 25 μl of 40 mg/ml R6G was added to the PLGAsolution before adding the protein. The solution was emulsified by probesonication for 20 sec (Duty cycle 20%, output control 3) to obtain w/oemulsion.

The resulting w/o emulsion was divided into two 18×150 mm disposableglass tubes and 12.5 ml of 2% PVA solution was added to each tube. Thesolution was emulsified by probe sonication for 30 sec (Duty cycle 20%,output control 3) to obtain the final w/o/w emulsion.

The final w/o/w was then combined in a 50 ml glass beaker and stirreduncovered for 20 hours with a magnetic stirrer at 400 rpm at 4° C. toallow solvent evaporation.

The solution was added to a 40 ml Oakridge tube and centrifuged at 3800rpm for 30 min. The supernatant was discarded and the pellet wasresuspended gently in 20 ml of distilled water. The washing step wasrepeated with two 20 min and one 15 min centrifugation. The supernatantwas discarded.

The resulting nanoparticle pellet was frozen in liquid nitrogen andlyophilized overnight at −88° C., 0.006 Torr.

The final product was stored at 4° C. and kept dry with Dry-rite calciumsulfate pellets till ready to use.

The morphology of the protein-loaded nanoparticles was visualized byScanning Electron Microscopy (SEM). A very small amount of nanoparticleswere placed on a double-sided adhesive tape attached to an aluminum stuband sputter coated with gold/palladium beam for 2 min. The coatedsampled were imaged with Philips XL30-FEG SEM at 10 kV.

The particle size of the nanoparticles was measured with ImageJ softwareusing the obtained SEM images. The diameter of approximately 150nanoparticles was measured, and the size distribution was plotted usingPrism software.

Total protein loading was estimated using BCA assay. Approximately 5-8mg of freeze-dried nanoparticles were accurately measured and added to 2ml of 5% SDS in 0.1 M NaOH solution and incubated with shaking for 24hours at room temperature until a clear solution was obtained (Rafati etal., 1997). The protein content was measured in triplicates for eachsample using BCA protein assay. The protein loading (%, w/w) wasexpressed as the amount of protein relative to the weight of thenanoparticles assayed (Coombes et al., 1998).

In vitro, the nanoparticles with the HA-HT-CPE protein were readilytaken up by GFP-Claudin4 CHO transfectants, showing both the function ofthe targeting peptide and the accessibility of the functional targetingpeptide in the nanoparticles.

In vitro uptake studies of R6G-labeled protein-loaded nanoparticles wereperformed in Green Fluorescence Protein (GFP) tagged claudin-4(GFP-Cldn-4) transfected Chinese Hamster Ovary (CHO) cells (Ling et al.,2008). Cells were maintained in F-12 Kaighn's medium supplemented with10% Fetal Bovine Serum and 0.8 mg/ml geneticin. The confocal studieswere performed in 50% confluent cells plated on cover slides placed in6-well plates, grown at 37° C. in 5% CO₂ incubator for 48 hrs. The cellswere washed with 2 ml of PBS and the medium was replaced by 1 ml of 10μg/ml nanoparticle solution (10 μg of protein/well), prepared bydissolving R6G-labled protein-loaded nanoparticles in culture mediumpre-warmed to 37° C. The cells were incubated at 37° C. in 5% CO₂incubator for one or two hrs. Upon incubation, cells were washed threetimes with 2 ml of PBS to remove unbound nanoparticles. Cells were thenfixed with 2 ml of 4% paraformaldehyde in PBS for 20 minutes at roomtemperature and washed with 2 ml of PBS+0.1% Tween20 for 3-5 min for twotimes. The cells on the cover slides were then mounted on glass slideswith Prolong Gold antifade reagent with DAPI and incubated for 24 hrs atroom temperature. Cells were analyzed using a BD CARV II spinning discconfocal microscope, using IPLab software. (see, e.g., FIG. 15).

In vivo, the same nanoparticles are also taken up in mucosal lymphoidtissues such as NALT and Peyer's patches, with a clear preference forthe HA-HT-CPE targeted nanoparticles. Interestingly, the enhanced uptakeis more evident for Peyer's patch, where the slower transit time of theintestinal contents may allow for the effect of the targeting peptide onM cell uptake. (See, e.g., FIG. 16).

1. A substantially purified peptide consisting of a sequence selectedfrom the group consisting of: (a) NSSYSGNYPYSILFQKF; (SEQ ID NO: 5)(b) SSYSGNYPYSIL; (SEQ ID NO: 6) (c) NSSYSGNYYSIL; and (SEQ ID NO: 7)(d) APWTEHSYYLSL (SEQ ID NO: 10).


2. The substantially purified peptide of claim 1, wherein the peptidebinds to a claudin-4 polypeptide.
 3. The substantially purified peptideof claim 1 further comprising a moiety of interest linked to thepeptide.
 4. The substantially purified peptide of claim 3, wherein themoiety of interest is a small molecule drug, a polypeptide or peptide,an antibody, a peptidomimetic, or a nanoparticle.
 5. The substantiallypurified peptide of claim 4, wherein the polypeptide or peptide is animmunogenic polypeptide or peptide.
 6. The substantially purifiedpeptide of claim 4, wherein the polypeptide or peptide is a therapeuticpolypeptide or peptide.
 7. The substantially purified peptide of claim4, wherein the polypeptide or peptide is a growth factor.
 8. Thesubstantially purified peptide of claim 4, wherein the small moleculedrug is an anticancer drug.
 9. The substantially purified peptide ofclaim 4, wherein the nanoparticle is a metallic nanoparticle.
 10. Thesubstantially purified peptide of claim 4, wherein the nanoparticle is abiocompatible polymer.
 11. The substantially purified peptide of claim10, wherein the biocompatible polymer is poly(lactide-co-glycolide)(PLGA).
 12. The substantially purified peptide of claim 11, wherein thenanoparticle comprising PLGA further comprises a therapeutic agent. 13.A substantially purified peptide consisting of a sequence selected fromthe group consisting of: (a) NSSYSGNYPYSILFQKF; (SEQ ID NO: 5)(b) SSYSGNYPYSIL; (SEQ ID NO: 6) (c) NSSYSGNYYSIL; and (SEQ ID NO: 7)(d) APWTEHSYYLSL (SEQ ID NO: 10),

wherein the peptide comprises at least one D-amino acid.
 14. Apharmaceutical composition comprising the peptide of claim 1 or
 13. 15.The composition of claim 14, in a controlled release formulation, in aliposomal form, in a lyophilized form or in a unit dosage form.
 16. Afusion polypeptide comprising the peptide of claim 1 further comprisinga polypeptide of interest linked to the peptide.
 17. The fusionpolypeptide of claim 16, wherein the polypeptide of interest comprisesan immunogenic molecule.
 18. The fusion peptide of claim 16, wherein thepeptide and the polypeptide of interest are separated by a peptidelinker.
 19. A method of modulating inflammation, asthma, allergy, cellproliferative disorders, metastasis of cancer cells, ion transportdisorders, magnesium transport defects in the kidney, inflammatory boweldisease, Clostridium perfringens enterotoxin (CPE) infection, myelinsheath formation disorder, multiple sclerosis (MS), autoimmuneencephalomyelitis, optic neuritis, and progressive multifocalleukoencephalopathy (PML), the method comprising administering to asubject the peptide of claim 1 or 13, or a fusion polypeptide of claim16 either alone or optionally with a pharmaceutically acceptablecarrier.
 20. A method of targeting a therapeutic to mucosal M cellscomprising linking a therapeutic moiety to the peptide of claim 1, 13,or a fusion polypeptide of claim 16 and contacting a mucosal M cell withthe peptide or fusion polypeptide.
 21. The method of claim 20, whereinthe mucosal M cell is in vivo.
 22. The method of claim 20, wherein thetherapeutic moiety is a cytotoxic drug, an immunopotentiating drug, aninhibitory nucleic acid molecule, a peptide, a polypeptide or apeptidomimetic.